Primer design

Two PCR primer sets were designed from the nucleotide sequences of the PRV-2 genome segments. For conventional RT–PCR, the primers lambda1-F (5´-GGTGAAGTTCATTCTTGCCAATC-3´) and lambda1-R (5´-ATATCCCGAAGCCTGACAGTCA-3´) were designed based on the nucleotide sequence of the helicase gene (segment L1), and amplified a 300-bp fragment. For RT–qPCR, the primers lambda2-F (5´-CGCTCCTCCAGCAACGAT-3´) and lambda2-R (5´-GGTGGATTGAGGCAGAGTTTG-3´) were designed to amplify a 55-bp fragment of the guanylyltransferase gene (segment L2). A commercial real-time primer for λpolyA (Takara Bio) was used to quantify the cDNA of the external control. The DNA fragments amplified with the lambda1-F/R or λpolyA primers were inserted into the pCR2.1 plasmid vector to generate the standards for quantification.